Modification of polypeptides

ABSTRACT

The invention provides a method for conjugating a peptide displayed on a genetic display system to a molecular scaffold performed on an ion exchange resin.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

All documents cited or referenced herein (“herein cited documents”), andall documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

FIELD OF THE INVENTION

The present invention concerns methods for production of polypeptideligands having a desired binding activity. In particular, the inventionconcerns the production of polypeptides which are covalently bound tomolecular scaffolds such that two or more peptide loops are subtendedbetween attachment points to the scaffold. Attachment of the molecularscaffold to the polypeptide is performed on a purification resin, whichcan take the form of magnetic resin beads.

BACKGROUND OF THE INVENTION

Cyclic peptides are able to bind with high affinity and targetspecificity to protein targets and hence are an attractive moleculeclass for the development of therapeutics. In fact, several cyclicpeptides are successfully used in the clinic, as for example theantibacterial peptide vancomycin, the immunosuppressant drugcyclosporine or the anti-cancer drug ocreotide (Driggers, et al., NatRev Drug Discov 2008, 7 (7), 608-24). Good binding properties resultfrom a relatively large interaction surface formed between the peptideand the target as well as the reduced conformational flexibility of thecyclic structures. Typically, macrocycles bind to surfaces of severalhundred square angstrom, as for example the cyclic peptide CXCR4antagonist CVX15 (400 Å²; Wu, B., et al., Science 330 (6007), 1066-71),a cyclic peptide with the Arg-Gly-Asp motif binding to integrin αVb3(355 Å²) (Xiong, J. P., et al., Science 2002, 296 (5565), 151-5) or thecyclic peptide inhibitor upain-1 binding to urokinase-type plasminogenactivator (603 Å²; Zhao, G., et al., J Struct Biol 2007, 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexiblethan linear peptides, leading to a smaller loss of entropy upon bindingto targets and resulting in a higher binding affinity. The reducedflexibility also leads to locking target-specific conformations,increasing binding specificity compared to linear peptides. This effecthas been exemplified by a potent and selective inhibitor of matrixmetalloproteinase 8, MMP-8) which lost its selectivity over other MMPswhen its ring was opened (Cherney, R. J., et al., J Med Chem 1998, 41(11), 1749-51). The favorable binding properties achieved throughmacrocyclization are even more pronounced in multicyclic peptides havingmore than one peptide ring as for example in vancomycin, nisin oractinomycin.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp, D. S, andMcNamara, P. E., J. Org. Chem, 1985; Timmerman, P. et al., ChemBioChem,2005). Meloen and co-workers had used tris(bromomethyl)benzene andrelated molecules for rapid and quantitative cyclisation of multiplepeptide loops onto synthetic scaffolds for structural mimicry of proteinsurfaces (Timmerman, P. et al., ChemBioChem, 2005). Methods for thegeneration of candidate drug compounds wherein said compounds aregenerated by linking cysteine containing polypeptides to a molecularscaffold as for example tris(bromomethyl)benzene are disclosed in WO2004/077062 and WO 2006/078161.

WO2004/077062 discloses a method of selecting a candidate drug compound.In particular, this document discloses various scaffold moleculescomprising first and second reactive groups, and contacting saidscaffold with a further molecule to form at least two linkages betweenthe scaffold and the further molecule in a coupling reaction.

WO2006/078161 discloses binding compounds, immunogenic compounds andpeptidomimetics. This document discloses the artificial synthesis ofvarious collections of peptides taken from existing proteins. Thesepeptides are then combined with a constant synthetic peptide having someamino acid changes introduced in order to produce combinatoriallibraries. By introducing this diversity via the chemical linkage toseparate peptides featuring various amino acid changes, an increasedopportunity to find the desired binding activity is provided. FIG. 1 ofthis document shows a schematic representation of the synthesis ofvarious loop peptide constructs. The constructs disclosed in thisdocument rely on —SH functionalised peptides, typically comprisingcysteine residues, and heteroaromatic groups on the scaffold, typicallycomprising benzylic halogen substituents such as bis- ortris-bromophenylbenzene. Such groups react to form a 3yclized3s linkagebetween the peptide and the scaffold.

Heinis at al. recently developed a phage display-based combinatorialapproach to generate and screen large libraries of bicyclic peptides totargets of interest (Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7;see also international patent application WO2009/098450). Briefly,combinatorial libraries of linear peptides containing three cysteineresidues and two regions of six random amino acids(Cys-(Xaa)₆-Cys-(Xaa)₆-Cys) were displayed on phage and 3yclized bycovalently linking the cysteine side chains to a small molecule(tris-(bromomethyl)benzene). Bicyclic peptides isolated in selectionsfor affinity to the human proteases cathepsin G and plasma Kallikrein(PK) had nanomolar inhibitory constants. The best inhibitor, PK15,inhibits human PK (Hpk) with a K_(i) of 3 Nm. Similarities in the aminoacid sequences of several isolated bicyclic peptides suggested that bothpeptide loops contribute to the binding. PK15 did not inhibit rat PK(81% sequence identity) nor the homologous human serine proteases factorXia (hfXla; 69% sequence identity) or thrombin (36% sequence identity)at the highest concentration tested (10 μM) (Heinis, et al., Nat ChemBiol 2009, 5 (7), 502-7). This finding suggested that the bicyclicinhibitor possesses high affinity for its target, and is highlyspecific.

Although the method disclosed by Heinis et al. is effective for themodification of displayed polypeptide ligands to produce bicyclicpeptides, its efficiency is very low. For example, infective phage aregenerated at a rate of only 1 in 350 per starting phage particle. Thepresent invention, therefore, developed an improved protocol for themodification of polypeptide ligands displayed on genetic displaysystems.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a method forconjugating a peptide displayed on a genetic display system to amolecular scaffold, comprising the steps of:

(a) combining polypeptides displayed on a genetic display system with apurification resin such that the display system is bound to the resinand treating the bound display system with a reducing agent;

(b) exposing the bound display system to the molecular scaffold;

(c) removing unreacted molecular scaffold from the bound display system;and

(d) eluting the display system from the purification resin.

The original method by Heinis et al. performed the conjugation ofpeptide and molecular scaffold (TBMB) in free solution. Phage, bearingpeptides which were (or were not) conjugated to the TBMB scaffold werethen isolated by centrifugation. The present invention obtained improvedresults by conjugating the phage to a solid phase purification resin,which can then be used to isolate the phage. For example, the resin canbe isolated by centrifugation or retained in columns; in a preferredembodiment, the resin is magnetic and can be isolated by the applicationof a magnetic field.

In embodiments, the genetic display system is selected from phagedisplay, ribosome display, mRNA display, yeast display and bacterialdisplay. In one embodiment, the genetic display system is phage display.

In one embodiment, step (a) is followed by a washing step beforeaddition of the molecular scaffold. Washing can be performed, forexample, with a solution of a reducing agent, for example the reducingagent used in step (a). Advantageously, the reducing agent used in thewashing step is less powerful or more dilute than the reducing agentused in step (a).

The resin-bound polypeptides may be exposed to the reducing agent inpurified form, or can be present in culture. Genetic display systemsinvolve replication in cells, such as bacteria or yeast; these cells maybe removed by purification, in which case step (a) can comprise awashing step, in which polypeptides bound to resin are washed in bufferand separated from the cell culture contaminants.

A suitable reducing agent is TCEP. Other reducing agents, such as DTT,can be used as set forth herein.

The reduction and conjugation reactions are preferably conducted at roomtemperature, such as 25° C. In the aforementioned method of Heinis etal., reactions are conducted at temperatures above room temperature, forexample 42° C.

The polypeptide is preferably a polypeptide which comprises at leastthree reactive groups, separated by at least two sequences which canform the “loops” of the polypeptide once conjugated to the molecularscaffold. The loops may be any suitable length, such as two, three,four, five, six, seven or more amino acids long. The loops may be thesame length, or different. Preferably, at least two loops are provided.In some embodiments, three, four, five, six or more loops may bepresent.

Reactive groups in the polypeptide are capable of forming covalentlinkages with the scaffold. Most commonly, reactive groups comprisecysteine residues.

Peptides are combined with a purification resin, which can be anysuitable resin which is useful as a solid phase for the purification ofprotein material. Many resins, such as ion-exchange resins includingbeads and chromatography materials are known in the art which are usefulfor this purpose.

In an advantageous embodiment, the resin is a magnetic resin, whichallows magnetic separation of the polypeptides bound to the geneticdisplay system.

The scaffold may be any structure which provides multiple attachmentpoints for the reactive groups of the polypeptide. Exemplary scaffoldsare described below. Scaffold molecules are conjugated to thepolypeptide whilst the polypeptides are incorporated into the geneticdisplay system, such that the genetic display system displays thepolypeptide ligand including the molecular scaffold. Excess scaffold isremoved.

After the scaffold has been conjugated to the polypeptides, the geneticdisplay systems incorporating the polypeptide ligands are eluted fromthe resin. The polypeptides can then be displayed on the genetic displaysystem in conjugated form, and selected by known means.

In embodiments, the polypeptide ligands are multispecific. In a firstconfiguration, for example, the polypeptide loops formed by theinteraction of the polypeptide with the molecular scaffold are capableof binding to more than one target. Within this configuration, in oneembodiment loops may be selected individually for binding to the desiredtargets, and then combined. In another embodiment, the loops areselected together, as part of a single structure, for binding todifferent desired targets.

In a second configuration, a functional group may be attached to the Nor C terminus, or both, of the polypeptide. The functional group maytake the form of a binding group, such as a polypeptide, including anantibody domain, an Fc domain or a further structured peptide asdescribed above, capable of binding to a target. It may moreover takethe form of a reactive group, capable of chemical bonding with a target.Moreover, it can be an effector group, including large plasma proteins,such as serum albumin, and a cell penetrating peptide.

In a third configuration, a functional group may be attached to themolecular scaffold itself. Examples of functional groups are as for thepreceding configuration.

In further embodiments, the polypeptide ligand comprises a polypeptidelinked to a molecular scaffold at n attachment points, wherein saidpolypeptide is cyclised and forms n separate loops subtended betweensaid n attachment points on the molecular scaffold, wherein n is greaterthan or equal to 2.

The polypeptide is preferably cyclised by N- to C-terminal fusion, andcan be cyclised before or after attachment to the molecular scaffold.Attachment before cyclisation is preferred.

Several methods are known in the art for peptide cyclisation. Forexample, the polypeptide is cyclised by N—C crosslinking, using acrosslinking agent such as EDC.

In another embodiment, the peptide can be designed to comprise aprotected N^(α) or C^(α) derivatised amino acid, and cyclised bydeprotection of the protected N^(α) or C^(α) derivatised amino acid tocouple said amino acid to the opposite terminus of the polypeptide.

In a preferred embodiment, the polypeptide is cyclised by enzymaticmeans.

For example, the enzyme is a transglutaminase, for instance a microbialtransglutaminase, such as Streptomyces mobaraensis transglutaminase. Inorder to take advantage of enzymatic cyclisation, it may be necessary toincorporate an N- and/or C-terminal substrate sequence for the enzyme inthe polypeptide. Some or all of the substrate sequence(s) can beeliminated during the enzymatic reaction, meaning that the cyclisedpolypeptide may not comprise the substrate sequences in its finalconfiguration.

In a still further embodiment, the polypeptide ligands according to theinvention are specific for human Kallikrein, and comprise a polypeptidecomprising at least three reactive groups, separated by at least twoloop sequences, and a molecular scaffold which forms covalent bonds withthe reactive groups of the polypeptide such that at least twopolypeptide loops are formed on the molecular scaffold, wherein theloops of the peptide ligand comprise three, four or five, but less thansix, amino acids.

Surprisingly, the present invention found that peptides comprising lessthan 6 amino acids in each loop can have a much higher binding affinityfor Kallikrein.

In one embodiment, the loops of the peptide ligand comprise three aminoacids and the polypeptide has the consensus sequenceG_(r)FxxG_(r)RVxG_(r), wherein G_(r) is a reactive group.

In another embodiment, the loops of the peptide ligand comprise fiveamino acids and a first loop comprises the consensus sequenceG_(r)GGxxNG_(r), wherein G_(r) is a reactive group. For example, twoadjacent loops of the polypeptide may comprise the consensus sequenceG_(r)GGxxNG_(r)RxxxxG_(r).

In one embodiment, the loops of the peptide ligand comprise five aminoacids and a first loop comprises the motifG_(r)x^(w)/_(F)Px^(K)/_(R)G_(r), wherein G_(r) is a reactive group. Inthe present context, the reference to a “first” loop does notnecessarily denote a particular position of the loop in a sequence. Insome embodiments, however, the first loop may be proximal loop in anamino terminus to carboxy terminus peptide sequence. For example, thepolypeptide further comprises a second, distal loop which comprises themotif G_(r) ^(T)/_(L)H^(Q)/_(T)xLG_(r). Examples of sequences of thefirst loop include G_(r)xWPARG_(r), G_(r)xWPSRG_(r), G_(r)xFPFRG_(r) andG_(r)xFPYRG_(r). In these examples, x may be any amino acid, but is forexample S or R.

In one embodiment, the loops of the peptide ligand comprise five aminoacids and a first loop comprises the motif G_(r)xHxDLG_(r), whereinG_(r) is a reactive group.

In one embodiment, the loops of the peptide ligand comprise five aminoacids and a first loop comprises the motif G_(r)THxxLG_(r), whereinG_(r) is a reactive group.

In one embodiment, the polypeptide comprises two adjacent loops whichcomprise the motif G_(r)x^(w)/_(F)Px^(K)/_(R)G_(r)^(T)/_(L)H^(Q)/_(T)DLG_(r).

In the examples herein, numbering refers to the positions in the loops,and ignores the reactive groups. Thus, inG_(r)x^(w)/_(F)Px^(K)/_(R)G_(r) ^(T)/_(L)H^(Q)/_(T)DLG_(r), x is inposition 1 and ^(T)/_(L) in position 6.

In the foregoing embodiments, the reactive group is preferably areactive amino acid. Preferably, the reactive amino acid is cysteine.

Variants of the polypeptides according to this aspect of the inventioncan be prepared as described above, by identifying those residues whichare available for mutation and preparing libraries which includemutations at those positions.

In a further aspect, there is provided a polypeptide ligand according tothe preceding aspect of the invention, which comprises one or morenon-natural amino acid substituents and is resistant to proteasedegradation.

The present invention found that certain non-natural amino acids permitbinding to plasma Kallikrein with nM Ki, whilst increasing residencetime in plasma significantly.

In one embodiment, the non-natural amino acid is selected from N-methylArginine, homoarginine and hydroxyproline. Preferably, N-methyl andhomo-derivatives of Arginine are used to replace Arginine, and proline 3can be preferably replaced by hydroxyproline, azetidine carboxylic acid,or an alpha-substituted amino acid, such as aminoisobutyric acid. Inanother embodiment, arginine may be replaced withguanidyl-phenylalanine.

In one embodiment, the polypeptide comprises a first loop whichcomprises the motif G_(r)xWPARG_(r), wherein P is replaced withazetidine carboxylic acid; and/or R is replaced with N-methyl arginine;and/or R is replaced with homoarginine; and/or R is replaced withguanidyl-phenylalanine.

In one embodiment, the polypeptide comprises a first loop whichcomprises the motif G_(r)xFPYRG_(r), wherein R is replaced with N-methylarginine; and/or R is replaced with homoarginine, and wherein proline isreplaced by azetidine carboxylic acid; and/or R is replaced withguanidyl-phenylalanine.

In one embodiment, the polypeptide ligand may further comprise asarcosine polymer, used as a linker to link polypeptide ligandstogether, or to attach one or more functional groups.

In some embodiments, the polypeptide ligand may be protease resistant.Protease resistant conjugates can be selected by screening a repertoireof polypeptide ligands for protease resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Assessment of the reaction conditions for linking phagedisplayed peptides to tris-(bromomethyl)benzene (TBMB). (A) Molecularmass of the GCGSGCGSGCG-D1-D2 fusion protein before and after reactionwith 10 μM TBMB in 20 mM NH₄HCO₃, 5 mM EDTA, pH 8, 20% ACN at 30° C. for1 hour determined by mass spectrometry. The mass difference of thereacted and non-reacted peptide fusion protein corresponds to the massof the small molecule core mesitylene. (C) Titres (transducing units) ofphage reduced and treated with various concentrations of TBMB in 20 mMNH₄HCO₃, 5 mM EDTA, pH 8, 20% ACN at 30° C. for 1 hour. Titres of phagefrom fdg3p0ss21 (black) and from library 1 (white) are shown.

FIG. 2: Chemical reaction of the tri-functional compound TBMB withpeptides containing one or two cysteines. (A) Plausible reactionmechanism of TBMB with a peptide fusion protein containing two cysteineresidues. (B) Mass spectra of a peptide fusion proteins with twocysteines before and after reaction with TBMB. (C) Plausible reactionmechanism of TBMB with a peptide fusion protein containing one cysteineresidue. (D) Mass spectra of a peptide fusion proteins with one cysteinebefore and after reaction with TBMB.

FIG. 3: The binding of resin-processed modified polypeptide ligands tokallikrein is illustrated.

FIG. 4: The effect of different buffers on the performance of themodification procedure. (A) the effect of different modificationbuffers, NaHCO3 and NH4CO3. (B) The effect of different concentrationsof NaCl elution buffer at different pH. (C) The effect of differentconcentrations of NaCl elution buffer and pH on elution in first andsecond steps in a two-step elution procedure.

FIG. 5: Target binding assay from the eluates of different samplestreated with different buffers and eluted at different pH.

FIG. 6: Illustration of quick and long magnetic modification protocols.

FIG. 7: Comparison of quick and long protocols for modification ofPK15-bearing phage: (A) comparison of phage titre by qPCR, and (B)functional comparison for Kallikrein binding.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry. Standardtechniques are used for molecular biology, genetic and biochemicalmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)ed., John Wiley & Sons, Inc.), which are incorporated herein byreference.

A (poly)peptide ligand or (poly)peptide conjugate, as referred toherein, refers to a polypeptide covalently bound to a molecularscaffold. Typically, such polypeptides comprise two or more reactivegroups which are capable of forming covalent bonds to the scaffold, anda sequence subtended between said reactive groups which is referred toas the loop sequence, since it forms a loop when the peptide is bound tothe scaffold. In the present case, the polypeptides comprise at leastthree reactive groups, and form at least two loops on the scaffold.

The reactive groups are groups capable of forming a covalent bond withthe molecular scaffold. Typically, the reactive groups are present onamino acid side chains on the peptide. Examples are amino-containinggroups such as cysteine, lysine and selenocysteine.

Specificity, in the context herein, refers to the ability of a ligand tobind or otherwise interact with its cognate target to the exclusion ofentities which are similar to the target. For example, specificity canrefer to the ability of a ligand to inhibit the interaction of a humanenzyme, but not a homologous enzyme from a different species. Using theapproach described herein, specificity can be modulated, that isincreased or decreased, so as to make the ligands more or less able tointeract with homologues or paralogues of the intended target.Specificity is not intended to be synonymous with activity, affinity oravidity, and the potency of the action of a ligand on its target (suchas, for example, binding affinity or level of inhibition) are notnecessarily related to its specificity.

Binding activity, as used herein, refers to quantitative bindingmeasurements taken from binding assays, for example as described herein.Therefore, binding activity refers to the amount of peptide ligand whichis bound at a given target concentration.

Multispecificity is the ability to bind to two or more targets.Typically, binding peptides are capable of binding to a single target,such as an epitope in the case of an antibody, due to theirconformational properties. However, peptides can be developed which canbind to two or more targets; dual specific antibodies, for example. Inthe present invention, the peptide ligands can be capable of binding totwo or more targets and are therefore be multispecific. Preferably, theybind to two targets, and are dual specific. The binding may beindependent, which would mean that the binding sites for the targets onthe peptide are not structurally hindered by the binding of one or otherof the targets. In this case both targets can be bound independently.More generally it is expected that the binding of one target will atleast partially impede the binding of the other.

A target is a molecule or part thereof to which the peptide ligands bindor otherwise interact with. Although binding is seen as a prerequisiteto activity of most kinds, and may be an activity in itself, otheractivities are envisaged. Thus, the present invention does not requirethe measurement of binding directly or indirectly.

The molecular scaffold is any molecule which is able to connect thepeptide at multiple points to impart one or more structural features tothe peptide. It is not a cross-linker, in that it does not merelyreplace a disulphide bond; instead, it provides two or more attachmentpoints for the peptide. Preferably, the molecular scaffold comprises atleast three attachment points for the peptide, referred to as scaffoldreactive groups. These groups are capable of reacting to the reactivegroups on the peptide to form a covalent bond. Preferred structures formolecular scaffolds are described below.

Screening for binding activity (or any other desired activity) isconducted according to methods well known in the art, for instance fromphage display technology. For example, targets immobilised to a solidphase can be used to identify and isolate binding members of arepertoire. Screening allows selection of members of a repertoireaccording to desired characteristics.

The term library refers to a mixture of heterogeneous polypeptides ornucleic acids. The library is composed of members, which are notidentical. To this extent, library is synonymous with repertoire.Sequence differences between library members are responsible for thediversity present in the library. The library may take the form of asimple mixture of polypeptides or nucleic acids, or may be in the formof organisms or cells, for example bacteria, viruses, animal or plantcells and the like, transformed with a library of nucleic acids.Preferably, each individual organism or cell contains only one or alimited number of library members.

In one embodiment, the nucleic acids are incorporated into expressionvectors, in order to allow expression of the polypeptides encoded by thenucleic acids. In a preferred aspect, therefore, a library may take theform of a population of host organisms, each organism containing one ormore copies of an expression vector containing a single member of thelibrary in nucleic acid form which can be expressed to produce itscorresponding polypeptide member. Thus, the population of host organismshas the potential to encode a large repertoire of genetically diversepolypeptide variants.

In one embodiment, a library of nucleic acids encodes a repertoire ofpolypeptides. Each nucleic acid member of the library preferably has asequence related to one or more other members of the library. By relatedsequence is meant an amino acid sequence having at least 50% identity,for example at least 60% identity, for example at least 70% identity,for example at least 80% identity, for example at least 90% identity,for example at least 95% identity, for example at least 98% identity,for example at least 99% identity to at least one other member of thelibrary. Identity can be judged across a contiguous segment of at least3 amino acids, for example at least 4, 5, 6, 7, 8, 9 or 10 amino acids,for example least 12 amino acids, for example least 14 amino acids, forexample least 16 amino acids, for example least 17 amino acids or thefull length of the reference sequence.

A repertoire is a collection of variants, in this case polypeptidevariants, which differ in their sequence. Typically, the location andnature of the reactive groups will not vary, but the sequences formingthe loops between them can be randomised. Repertoires differ in size,but should be considered to comprise at least 10² members. Repertoiresof 10¹¹ or more members can be constructed.

A set of polypeptide ligands, as used herein, refers to a plurality ofpolypeptide ligands which can be subjected to selection in the methodsdescribed. Potentially, a set can be a repertoire, but it may also be asmall collection of polypeptides, from at least 2 up to 10, 20, 50, 100or more.

A group of polypeptide ligands, as used herein, refers to two or moreligands. In one embodiment, a group of ligands comprises only ligandswhich share at least one target specificity. Typically, a group willconsist of from at least 2, 3, 4, 5, 6, 7, 8, 9 or 10, 20, 50, 100 ormore ligands. In one embodiment, a group consists of 2 ligands.

In an embodiment of the present invention, provided is a method forconjugating a peptide displayed on a genetic display system to amolecular scaffold, comprising the steps of:

(a) combining polypeptides displayed on a genetic display system with apurification resin such that the display system is bound to the resinand treating the bound display system with a reducing agent;(b) exposing the bound display system to the molecular scaffold;(c) removing unreacted molecular scaffold from the bound display system;and(d) eluting the display system from the purification resin.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein the display system is phage display.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein step (a) is followed by a washing stepbefore addition of the molecular scaffold

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein the display system is washed in a dilutesolution of reducing agent.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein the wash solution further comprises achelating agent.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein the reducing agent is TCEP.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein the scaffold is TBMB.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein the molecular scaffold is added in thepresence of aqueous acetonitrile.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein the resin is an anion exchange resin.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein the resin is magnetic.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein step (a) is performed for 20 minutes at roomtemperature.

In another embodiment of the present invention, provided is the methodfor conjugating a peptide displayed on a genetic display system to amolecular scaffold, wherein step (b) is performed for 10 minutes at roomtemperature.

(i) Molecular Scaffold

Molecular scaffolds are described in, for example, WO2009098450 andreferences cited therein, particularly WO2004077062 and WO2006078161.

As noted in the foregoing documents, the molecular scaffold may be asmall molecule, such as a small organic molecule.

In one embodiment the molecular scaffold may be, or may be based on,natural monomers such as nucleosides, sugars, or steroids. For examplethe molecular scaffold may comprise a short polymer of such entities,such as a dimer or a trimer.

In one embodiment the molecular scaffold is a compound of knowntoxicity, for example of low toxicity. Examples of suitable compoundsinclude cholesterols, nucleotides, steroids, or existing drugs such astamazepam.

In one embodiment the molecular scaffold may be a macromolecule. In oneembodiment the molecular scaffold is a macromolecule composed of aminoacids, nucleotides or carbohydrates.

In one embodiment the molecular scaffold comprises reactive groups thatare capable of reacting with functional group(s) of the polypeptide toform covalent bonds.

The molecular scaffold may comprise chemical groups as amines, thiols,alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters,alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkylhalides and acyl halides.

In one embodiment, the molecular scaffold may comprise or may consist oftris(bromomethyl)benzene, especially 1,3,5-Tris(bromomethyl)benzene(‘TBMB’), or a derivative thereof.

In one embodiment, the molecular scaffold is2,4,6-Tris(bromomethyl)mesitylene. It is similar to1,3,5-Tris(bromomethyl)benzene but contains additionally three methylgroups attached to the benzene ring. This has the advantage that theadditional methyl groups may form further contacts with the polypeptideand hence add additional structural constraint.

The molecular scaffold of the invention contains chemical groups thatallow functional groups of the polypeptide of the encoded library of theinvention to form covalent links with the molecular scaffold. Saidchemical groups are selected from a wide range of functionalitiesincluding amines, thiols, alcohols, ketones, aldehydes, nitriles,carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides,maleimides, azides, alkyl halides and acyl halides.

(ii) Polypeptide

The reactive groups of the polypeptides can be provided by side chainsof natural or non-natural amino acids. The reactive groups of thepolypeptides can be selected from thiol groups, amino groups, carboxylgroups, guanidinium groups, phenolic groups or hydroxyl groups. Thereactive groups of the polypeptides can be selected from azide,keto-carbonyl, alkyne, vinyl, or aryl halide groups. The reactive groupsof the polypeptides for linking to a molecular scaffold can be the aminoor carboxy termini of the polypeptide.

In some embodiments each of the reactive groups of the polypeptide forlinking to a molecular scaffold are of the same type. For example, eachreactive group may be a cysteine residue. Further details are providedin WO2009098450.

In some embodiments the reactive groups for linking to a molecularscaffold may comprise two or more different types, or may comprise threeor more different types. For example, the reactive groups may comprisetwo cysteine residues and one lysine residue, or may comprise onecysteine residue, one lysine residue and one N-terminal amine.

Cysteine can be employed because it has the advantage that itsreactivity is most different from all other amino acids. Scaffoldreactive groups that could be used on the molecular scaffold to reactwith thiol groups of cysteines are alkyl halides (or also namedhalogenoalkanes or haloalkanes). Examples are bromomethylbenzene (thescaffold reactive group exemplified by TBMB) or iodoacetamide. Otherscaffold reactive groups that are used to couple selectively compoundsto cysteines in proteins are maleimides. Examples of maleimides whichmay be used as molecular scaffolds in the invention include:tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene,tris-(maleimido)benzene. Selenocysteine is also a natural amino acidwhich has a similar reactivity to cysteine and can be used for the samereactions. Thus, wherever cysteine is mentioned, it is typicallyacceptable to substitute selenocysteine unless the context suggestsotherwise.

Lysines (and primary amines of the N-terminus of peptides) are alsosuited as reactive groups to modify peptides on phage by linking to amolecular scaffold. However, they are more abundant in phage proteinsthan cysteines and there is a higher risk that phage particles mightbecome cross-linked or that they might lose their infectivity.Nevertheless, it has been found that lysines are especially useful inintramolecular reactions (e.g. when a molecular scaffold is alreadylinked to the phage peptide) to form a second or consecutive linkagewith the molecular scaffold. In this case the molecular scaffold reactspreferentially with lysines of the displayed peptide (in particularlysines that are in close proximity). Scaffold reactive groups thatreact selectively with primary amines are succinimides, aldehydes oralkyl halides. In the bromomethyl group that is used in a number of theaccompanying examples, the electrons of the benzene ring can stabilizethe cationic transition state. This particular aryl halide is therefore100-1000 times more reactive than alkyl halides. Examples ofsuccinimides for use as molecular scaffold include tris-(succinimidylaminotriacetate), 1,3,5-Benzenetriacetic acid. Examples of aldehydes foruse as molecular scaffold include Triformylmethane. Examples of alkylhalides for use as molecular scaffold include1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene,1,3,5-Tris(bromomethyl)benzene,1,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene.

The amino acids with reactive groups for linking to a molecular scaffoldmay be located at any suitable positions within the polypeptide. Inorder to influence the particular structures or loops created, thepositions of the amino acids having the reactive groups may be varied bythe skilled operator, e.g. by manipulation of the nucleic acid encodingthe polypeptide in order to mutate the polypeptide produced. By suchmeans, loop length can be manipulated in accordance with the presentteaching.

For example, the polypeptide can comprise the sequenceAC(X)_(n)C(X)_(m)CG, wherein X stands for a random natural amino acid, Afor alanine, C for cysteine and G for glycine and n and m, which may bethe same or different, are numbers between 3 and 6.

(iii) Reactive Groups of the Polypeptide

The molecular scaffold of the invention may be bonded to the polypeptidevia functional or reactive groups on the polypeptide. These aretypically formed from the side chains of particular amino acids found inthe polypeptide polymer. Such reactive groups may be a cysteine sidechain, a lysine side chain, or an N-terminal amine group or any othersuitable reactive group. Again, details may be found in WO2009098450.

Examples of reactive groups of natural amino acids are the thiol groupof cysteine, the amino group of lysine, the carboxyl group of aspartateor glutamate, the guanidinium group of arginine, the phenolic group oftyrosine or the hydroxyl group of serine. Non-natural amino acids canprovide a wide range of reactive groups including an azide, aketo-carbonyl, an alkyne, a vinyl, or an aryl halide group. The aminoand carboxyl group of the termini of the polypeptide can also serve asreactive groups to form covalent bonds to a molecular scaffold/molecularcore.

The polypeptides of the invention contain at least three reactivegroups. Said polypeptides can also contain four or more reactive groups.The more reactive groups are used, the more loops can be formed in themolecular scaffold.

In a preferred embodiment, polypeptides with three reactive groups aregenerated. Reaction of said polypeptides with a molecularscaffold/molecular core having a three-fold rotational symmetrygenerates a single product isomer. The generation of a single productisomer is favourable for several reasons. The nucleic acids of thecompound libraries encode only the primary sequences of the polypeptidebut not the isomeric state of the molecules that are formed uponreaction of the polypeptide with the molecular core. If only one productisomer can be formed, the assignment of the nucleic acid to the productisomer is clearly defined. If multiple product isomers are formed, thenucleic acid cannot give information about the nature of the productisomer that was isolated in a screening or selection process. Theformation of a single product isomer is also advantageous if a specificmember of a library of the invention is synthesized. In this case, thechemical reaction of the polypeptide with the molecular scaffold yieldsa single product isomer rather than a mixture of isomers.

In another embodiment of the invention, polypeptides with four reactivegroups are generated. Reaction of said polypeptides with a molecularscaffold/molecular core having a tetrahedral symmetry generates twoproduct isomers. Even though the two different product isomers areencoded by one and the same nucleic acid, the isomeric nature of theisolated isomer can be determined by chemically synthesizing bothisomers, separating the two isomers and testing both isomers for bindingto a target ligand.

In one embodiment of the invention, at least one of the reactive groupsof the polypeptides is orthogonal to the remaining reactive groups. Theuse of orthogonal reactive groups allows the directing of saidorthogonal reactive groups to specific sites of the molecular core.Linking strategies involving orthogonal reactive groups may be used tolimit the number of product isomers formed. In other words, by choosingdistinct or different reactive groups for one or more of the at leastthree bonds to those chosen for the remainder of the at least threebonds, a particular order of bonding or directing of specific reactivegroups of the polypeptide to specific positions on the molecularscaffold may be usefully achieved.

In another embodiment, the reactive groups of the polypeptide of theinvention are reacted with molecular linkers wherein said linkers arecapable to react with a molecular scaffold so that the linker willintervene between the molecular scaffold and the polypeptide in thefinal bonded state.

In some embodiments, amino acids of the members of the libraries or setsof polypeptides can be replaced by any natural or non-natural aminoacid. Excluded from these exchangeable amino acids are the onesharbouring functional groups for cross-linking the polypeptides to amolecular core, such that the loop sequences alone are exchangeable. Theexchangeable polypeptide sequences have either random sequences,constant sequences or sequences with random and constant amino acids.The amino acids with reactive groups are either located in definedpositions within the polypeptide, since the position of these aminoacids determines loop size.

In one embodiment, an polypeptide with three reactive groups has thesequence (X)_(l)Y(X)_(m)Y(X)_(n)Y(X)_(o), wherein Y represents an aminoacid with a reactive group, X represents a random amino acid, m and nare numbers between 3 and 6 defining the length of interveningpolypeptide segments, which may be the same or different, and l and oare numbers between 0 and 20 defining the length of flanking polypeptidesegments.

Alternatives to thiol-mediated conjugations can be used to attach themolecular scaffold to the peptide via covalent interactions.Alternatively these techniques may be used in modification or attachmentof further moieties (such as small molecules of interest which aredistinct from the molecular scaffold) to the polypeptide after they havebeen selected or isolated according to the present invention—in thisembodiment then clearly the attachment need not be covalent and mayembrace non-covalent attachment. These methods may be used instead of(or in combination with) the thiol mediated methods by producing phagethat display proteins and peptides bearing unnatural amino acids withthe requisite chemical reactive groups, in combination small moleculesthat bear the complementary reactive group, or by incorporating theunnatural amino acids into a chemically or recombinantly synthesisedpolypeptide when the molecule is being made after theselection/isolation phase. Further details can be found in WO2009098450or Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7.

(iv) Combination of Loops to Form Multispecific Molecules

Loops from peptide ligands, or repertoires of peptide ligands, areadvantageously combined by sequencing and de novo synthesis of apolypeptide incorporating the combined loops. Alternatively, nucleicacids encoding such polypeptides can be synthesised.

Where repertoires are to be combined, particularly single looprepertoires, the nucleic acids encoding the repertoires areadvantageously digested and re-ligated, to form a novel repertoirehaving different combinations of loops from the constituent repertoires.Phage vectors can include polylinkers and other sites for restrictionenzymes which can provide unique points for cutting and relegation thevectors, to create the desired multispecific peptide ligands. Methodsfor manipulating phage libraries are well known in respect ofantibodies, and can be applied in the present case also.

(v) Attachment of Effector Groups and Functional Groups

Effector and/or functional groups can be attached, for example, to the Nor C termini of the polypeptide, or to the molecular scaffold.

Appropriate effector groups include antibodies and parts or fragmentsthereof. For instance, an effector group can include an antibody lightchain constant region (CL), an antibody CH1 heavy chain domain, anantibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, orany combination thereof, in addition to the one or more constant regiondomains. An effector group may also comprise a hinge region of anantibody (such a region normally being found between the CH1 and CH2domains of an IgG molecule).

In a further preferred embodiment of this aspect of the invention, aneffector group according to the present invention is an Fc region of anIgG molecule. Advantageously, a peptide ligand-effector group accordingto the present invention comprises or consists of a peptide ligand Fcfusion having a tβ half-life of a day or more, two days or more, 3 daysor more, 4 days or more, 5 days or more, 6 days or more or 7 days ormore. Most advantageously, the peptide ligand according to the presentinvention comprises or consists of a peptide ligand Fc fusion having atβ half-life of a day or more.

Functional groups include, in general, binding groups, drugs, reactivegroups for the attachment of other entities, functional groups which aiduptake of the macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate into cells will allow peptidesagainst intracellular targets to be effective. Targets that can beaccessed by peptides with the ability to penetrate into cells includetranscription factors, intracellular signalling molecules such astyrosine kinases and molecules involved in the apoptotic pathway.Functional groups which enable the penetration of cells include peptidesor chemical groups which have been added either to the peptide or themolecular scaffold. Peptides such as those derived from such as VP22,HIV-Tat, a homeobox protein of Drosophila (Antennapedia), e.g. asdescribed in Chen and Harrison, Biochemical Society Transactions (2007)Volume 35, part 4, p 821 “Cell-penetrating peptides in drug development:enabling intracellular targets” and “Intracellular delivery of largemolecules and small peptides by cell penetrating peptides” by Gupta etal. in Advanced Drug Discovery Reviews (2004) Volume 57 9637. Examplesof short peptides which have been shown to be efficient at translocationthrough plasma membranes include the 16 amino acid penetration peptidefrom Drosophila Antennapedia protein (Derossi et al (1994) J Biol. Chem.Volume 269 p 10444 “The third helix of the Antennapedia homeodomaintranslocates through biological membranes”), the 18 amino acid ‘modelamphipathic peptide’ (Oehlke et al (1998) Biochim Biophys Acts Volume1414 p 127 “Cellular uptake of an alpha-helical amphipathic modelpeptide with the potential to deliver polar compounds into the cellinterior non-endocytically”) and arginine rich regions of the HIV TATprotein. Non peptidic approaches include the use of small moleculemimics or SMOCs that can be easily attached to biomolecules (Okuyama etal (2007) Nature Methods Volume 4 p 153 ‘Small-molecule mimics of ana-helix for efficient transport of proteins into cells’. Other chemicalstrategies to add guanidinium groups to molecules also enhance cellpenetration (Elson-Scwab et al (2007) J Biol Chem Volume 282 p 13585“Guanidinylated Neomcyin Delivers Large Bioactive Cargo into cellsthrough a heparin Sulphate Dependent Pathway”). Small molecular weightmolecules such as steroids may be added to the molecular scaffold toenhance uptake into cells.

One class of functional groups which may be attached to peptide ligandsincludes antibodies and binding fragments thereof, such as Fab, Fv orsingle domain fragments. In particular, antibodies which bind toproteins capable of increasing the half life of the peptide ligand invivo may be used.

RGD peptides, which bind to integrins which are present on many cells,may also be incorporated.

In one embodiment, a peptide ligand-effector group according to theinvention has a tβ half-life selected from the group consisting of: 12hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 daysor more, 5 days or more, 6 days or more, 7 days or more, 8 days or more,9 days or more, 10 days or more, 11 days or more, 12 days or more, 13days or more, 14 days or more, 15 days or more or 20 days or more.Advantageously a peptide ligand-effector group or composition accordingto the invention will have a tβ half life in the range 12 to 60 hours.In a further embodiment, it will have at half-life of a day or more. Ina further embodiment still, it will be in the range 12 to 26 hours.

Functional groups include drugs, such as cytotoxic agents for cancertherapy. These include Alkylating agents such as Cisplatin andcarboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide,chlorambucil, ifosfamide; Anti-metabolites including purine analogsazathioprine and mercaptopurine)) or pyrimidine analogs; plant alkaloidsand terpenoids including vinca alkaloids such as Vincristine,Vinblastine, Vinorelbine and Vindesine; Podophyllotoxin and itsderivatives etoposide and teniposide; Taxanes, including paclitaxel,originally known as Taxol; topoisomerase inhibitors includingcamptothecins: irinotecan and topotecan, and type II inhibitorsincluding amsacrine, etoposide, etoposide phosphate, and teniposide.Further agents can include Antitumour antibiotics which include theimmunosuppressant dactinomycin (which is used in kidneytransplantations), doxorubicin, epirubicin, bleomycin and others.

Possible effector groups also include enzymes, for instance such ascarboxypeptidase G2 for use in enzyme/prodrug therapy, where the peptideligand replaces antibodies in ADEPT.

(vi) Peptide Modification

To develop the bicyclic peptides (Bicycles; peptides conjugated tomolecular scaffolds) into a suitable drug-like molecule, whether that befor injection, inhalation, nasal, ocular, oral or topicaladministration, a number of properties need considered. The following atleast need to be designed into a given lead Bicycle:

-   -   protease stability, whether this concerns Bicycle stability to        plasma proteases, epithelial (“membrane-anchored”) proteases,        gastric and intestinal proteases, lung surface proteases,        intracellular proteases and the like. Protease stability should        be maintained between different species such that a Bicycle lead        candidate can be developed in animal models as well as        administered with confidence to humans.    -   replacement of oxidation-sensitive residues, such as tryptophan        and methionine with oxidation-resistant analogues in order to        improve the pharmaceutical stability profile of the molecule    -   a desirable solubility profile, which is a function of the        proportion of charged and hydrophilic versus hydrophobic        residues, which is important for formulation and absorption        purposes    -   correct balance of charged versus hydrophobic residues, as        hydrophobic residues influence the degree of plasma protein        binding and thus the concentration of the free available        fraction in plasma, while charged residues (in particular        arginines) may influence the interaction of the peptide with the        phospholipid membranes on cell surfaces. The two in combination        may influence half-life, volume of distribution and exposure of        the peptide drug, and can be tailored according to the clinical        endpoint. In addition, the correct combination and number of        charged versus hydrophobic residues may reduce irritation at the        injection site (were the peptide drug administered        subcutaneously).    -   a tailored half-life, depending on the clinical indication and        treatment regimen. It may be prudent to develop an unmodified        molecule for short exposure in an acute illness management        setting, or develop a bicyclic peptide with chemical        modifications that enhance the plasma half-life, and hence be        optimal for the management of more chronic disease states.

Approaches to stabilise therapeutic peptide candidates againstproteolytic degradation are numerous, and overlap with thepeptidomimetics field (for reviews see Gentilucci et al, Curr.Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr.Medicinal Chem (2009), 16, 4399-418).

These include

-   -   Cyclisation of peptide    -   N- and C-terminal capping, usually N-terminal acetylation and        C-terminal amidation.    -   Alanine scans, to reveal and potentially remove the proteolytic        attack site(s).    -   D-amino acid replacement, to probe the steric requirements of        the amino acid side chain, to increase proteolytic stability by        steric hindrance and by a propensity of D-amino acids to        stabilise β-turn conformations (Tugyi at al (2005) PNAS, 102(2),        413-418).    -   N-methyl/N-alkyl amino acid replacement, to impart proteolytic        protection by direct modification of the scissile amide bond        (Fiacco et al, Chembiochem. (2008), 9(14), 2200-3).        N-methylation also has strong effect on the torsional angles of        the peptide bond, and is believed to aid in cell penetration &        oral availability (Biron at al (2008), Angew. Chem. Int. Ed.,        47, 2595-99)    -   Incorporation of non-natural amino acids, i.e. by employing        -   Isosteric/isoelectronic side chains that are not recognised            by proteases, yet have no effect on target potency        -   Constrained amino acid side chains, such that proteolytic            hydrolysis of the nearby peptide bond is conformationally            and sterically impeded. In particular, these concern proline            analogues, bulky sidechains, Cα-disubstituted derivatives            (where the simplest derivative is Aib, H₂N—C(CH₃)₂—COOH),            and cyclo amino acids, a simple derivative being            amino-cyclopropylcarboxylic acid).    -   Peptide bond surrogates, and examples include        -   N-alkylation (see above, i.e. CO—NR)        -   Reduced peptide bonds (CH₂—NH—)        -   Peptoids (N-alkyl amino acids, NR—CH₂—CO)        -   Thio-amides (CS—NH)        -   Azapeptides (CO—NH—NR)        -   Trans-alkene (RHC═C—)        -   Retro-inverso (NH—CO)        -   Urea surrogates (NH—CO—NHR)    -   Peptide backbone length modulation        -   i.e. β^(2/3)-amino acids, (NH—CR—CH₂—CO, NH—CH₂—CHR—CO),    -   Substitutions on the alpha-carbon on amino acids, which        constrains backbone conformations, the simplest derivative being        Aminoisobutyric acid (Aib).

It should be explicitly noted that some of these modifications may alsoserve to deliberately improve the potency of the peptide against thetarget, or, for example to identify potent substitutes for theoxidation-sensitive amino acids (Trp and Met).

(B) Repertoires, Sets and Groups of Polypeptide Ligands (i) Constructionof Libraries

Libraries intended for selection may be constructed using techniquesknown in the art, for example as set forth in WO2004/077062, orbiological systems, including phage vector systems as described herein.Other vector systems are known in the art, and include other phage (forinstance, phage lambda), bacterial plasmid expression vectors,eukaryotic cell-based expression vectors, including yeast vectors, andthe like. For example, see WO2009098450 or Heinis, et al., Nat Chem Biol2009, 5 (7), 502-7.

Non-biological systems such as those set forth in WO2004/077062 arebased on conventional chemical screening approaches. They are simple,but lack the power of biological systems since it is impossible, or atleast impracticably onerous, to screen large libraries of peptideligands. However, they are useful where, for instance, only a smallnumber of peptide ligands needs to be screened. Screening by suchindividual assays, however, may be time-consuming and the number ofunique molecules that can be tested for binding to a specific targetgenerally does not exceed 10⁶ chemical entities.

In contrast, biological screening or selection methods generally allowthe sampling of a much larger number of different molecules. Thusbiological methods can be used in application of the invention. Inbiological procedures, molecules are assayed in a single reaction vesseland the ones with favourable properties (i.e. binding) are physicallyseparated from inactive molecules. Selection strategies are availablethat allow to generate and assay simultaneously more than 10¹³individual compounds. Examples for powerful affinity selectiontechniques are phage display, ribosome display, mRNA display, yeastdisplay, bacterial display or RNA/DNA aptamer methods. These biologicalin vitro selection methods have in common that ligand repertoires areencoded by DNA or RNA. They allow the propagation and the identificationof selected ligands by sequencing. Phage display technology has forexample been used for the isolation of antibodies with very high bindingaffinities to virtually any target.

When using a biological system, once a vector system is chosen and oneor more nucleic acid sequences encoding polypeptides of interest arecloned into the library vector, one may generate diversity within thecloned molecules by undertaking mutagenesis prior to expression;alternatively, the encoded proteins may be expressed and selected beforemutagenesis and additional rounds of selection are performed.

Mutagenesis of nucleic acid sequences encoding structurally optimisedpolypeptides is carried out by standard molecular methods. Of particularuse is the polymerase chain reaction, or PCR, (Mullis and Faloona (1987)Methods Enzymol., 155: 335, herein incorporated by reference). PCR,which uses multiple cycles of DNA replication catalysed by athermostable, DNA-dependent DNA polymerase to amplify the targetsequence of interest, is well known in the art. The construction ofvarious antibody libraries has been discussed in Winter et al. (1994)Ann. Rev. Immunology 12, 433-55, and references cited therein.

Alternatively, given the short chain lengths of the polypeptidesaccording to the invention, the variants are preferably synthesised denovo and inserted into suitable expression vectors. Peptide synthesiscan be carried out by standard techniques known in the art, as describedabove. Automated peptide synthesisers are widely available, such as theApplied Biosystems ABI 433 (Applied Biosystems, Foster City, Calif.,USA)

(ii) Genetically Encoded Diversity

In one embodiment, the polypeptides of interest are genetically encoded.This offers the advantage of enhanced diversity together with ease ofhandling. An example of a genetically polypeptide library is a mRNAdisplay library. Another example is a replicable genetic display package(rgdp) library such as a phage display library. In one embodiment, thepolypeptides of interest are genetically encoded as a phage displaylibrary.

Thus, in one embodiment the complex of the invention comprises areplicable genetic display package (rgdp) such as a phage particle. Inthese embodiments, the nucleic acid can be comprised by the phagegenome. In these embodiments, the polypeptide can be comprised by thephage coat.

In some embodiments, the invention may be used to produce a geneticallyencoded combinatorial library of polypeptides which are generated bytranslating a number of nucleic acids into corresponding polypeptidesand linking molecules of said molecular scaffold to said polypeptides.

The genetically encoded combinatorial library of polypeptides may begenerated by phage display, yeast display, ribosome display, bacterialdisplay or mRNA display.

Techniques and methodology for performing phage display can be found inWO2009098450.

In one embodiment, screening may be performed by contacting a library,set or group of polypeptide ligands with a target and isolating one ormore member(s) that bind to said target.

In another embodiment, individual members of said library, set or groupare contacted with a target in a screen and members of said library thatbind to said target are identified.

In another embodiment, members of said library, set or group aresimultaneously contacted with a target and members that bind to saidtarget are selected.

The target(s) may be a peptide, a protein, a polysaccharide, a lipid, aDNA or a RNA.

The target may be a receptor, a receptor ligand, an enzyme, a hormone ora cytokine.

The target may be a prokaryotic protein, a eukaryotic protein, or anarcheal protein. More specifically the target ligand may be a mammalianprotein or an insect protein or a bacterial protein or a fungal proteinor a viral protein.

The target ligand may be an enzyme, such as a protease.

It should be noted that the invention also embraces polypeptide ligandsisolated from a screen according to the invention. In one embodiment thescreening method(s) of the invention further comprise the step of:manufacturing a quantity of the polypeptide isolated as capable ofbinding to said targets.

The invention also relates to peptide ligands having more than twoloops. For example, tricyclic polypeptides joined to a molecularscaffold can be created by joining the N- and C-termini of a bicyclicpolypeptide joined to a molecular scaffold according to the presentinvention. In this manner, the joined N and C termini create a thirdloop, making a tricyclic polypeptide. This embodiment need not becarried out on phage, but can be carried out on a polypeptide-molecularscaffold conjugate as described herein. Joining the N- and C-termini isa matter of routine peptide chemistry. In case any guidance is needed,the C-terminus may be activated and/or the N- and C-termini may beextended for example to add a cysteine to each end and then join them bydisulphide bonding. Alternatively the joining may be accomplished by useof a linker region incorporated into the N/C termini. Alternatively theN and C termini may be joined by a conventional peptide bond.Alternatively any other suitable means for joining the N and C terminimay be employed, for example N—C-cyclization could be done by standardtechniques, for example as disclosed in Linde et al. Peptide Science 90,671-682 (2008) “Structure-activity relationship and metabolic stabilitystudies of backbone cyclization and N-methylation of melanocortinpeptides”, or as in Hess et al. J. Med. Chem. 51, 1026-1034 (2008)“backbone cyclic peptidomimetic melanocortin-4 receptor agonist as anovel orally administered drug lead for treating obesity”. One advantageof such tricyclic molecules is the avoidance of proteolytic degradationof the free ends, in particular by exoprotease action. Another advantageof a tricyclic polypeptide of this nature is that the third loop may beutilised for generally applicable functions such as BSA binding, cellentry or transportation effects, tagging or any other such use. It willbe noted that this third loop will not typically be available forselection (because it is not produced on the phage but only on thepolypeptide-molecular scaffold conjugate) and so its use for other suchbiological functions still advantageously leaves both loops 1 and 2 forselection/creation of specificity.

(iii) Phage Purification

In accordance with the present invention, phage purification beforereaction with the molecular scaffold is optional. In the event thatpurification is desired, any suitable means for purification of thephage may be used. Standard techniques may be applied in the presentinvention. For example, phage may be purified by filtration or byprecipitation such as PEG precipitation; phage particles may be producedand purified by polyethylene-glycol (PEG) precipitation as describedpreviously. Details can be found in WO2009098450.

In case further guidance is needed, reference is made to Jespers et al(Protein Engineering Design and Selection 2004 17(10):709-713. Selectionof optical biosensors from chemisynthetic antibody libraries.) In oneembodiment phage may be purified as taught therein. The text of thispublication is specifically incorporated herein by reference for themethod of phage purification; in particular reference is made to thematerials and methods section starting part way down the right-column atpage 709 of Jespers et al.

Moreover, the phage may be purified as published by Marks et al J. Mol.Biol vol 222 pp 581-597, which is specifically incorporated herein byreference for the particular description of how the phageproduction/purification is carried out.

If phage purification is not desired, culture medium including phage canbe mixed directly with a purification resin and a reducing agent (suchas TCEP), as set forth in the examples herein.

(iv) Reaction Chemistry

In comparison to the conditions which are set out in WO2009098450 byHeinis et al., the reaction chemistry used in the present inventionprovides for a rapid and efficient generation of polypeptide ligandsdisplayed on phage. Reactions conditions used in the present inventionpreferably comprise the following steps, all preferably conducted atroom temperature:

-   -   1. Culture medium from which bacterial cells have been removed,        containing phage expressing the desired polypeptide(s), is mixed        with buffer, reducing agent and resin equilibrated in buffer.    -   2. The resin is isolated and resuspended in buffer and dilute        reducing agent.    -   3. The polypeptides are exposed to the molecular scaffold and        reacted therewith such that the molecular scaffold forms        covalent bonds with the polypeptide.    -   4. The samples are washed to remove excess unreacted scaffold.    -   5. Phage are eluted from the resin.

The buffer is preferably pH 8.0; it is not necessary to adjust buffer pHin the final solution. Suitable buffers include NaHCO₃, initially at pH8.0. Alternative buffers may be used, including buffers with a pH in thephysiological range, including NH₄CO₃, HEPES and Tris-hydroxymethylaminoethane, Tris, Tris-Acetate or MOPS. The NaHCO₃ buffer is preferablyused at a concentration of 1M, adding 1 ml to a suspension of resin toequilibrate the resin.

The resin is preferably an ion exchange resin. Ion exchange resins areknown in the art, and include any material suitable for anion exchangechromatography known in the art, such as an agarose based chromatographymaterial, e.g. sepharoses like Fast Flow or Capto, polymeric syntheticmaterial, e.g. a polymethacrylate such as Toyopearls,polystyrene/divinylbenzene, such as Poros, Source, or cellulose, e.g.Cellufine. In a preferred embodiment, the anion exchange resin materialincludes, but is not limited to a resin that carries a primary amine asligand, e.g. aminohexyl sepharose, benzamidine sepharose, lysinesepharose, or arginine sepharose. In another preferred embodiment, theanion exchange resin material includes, but is not limited to a resinhaving a positively charged moiety at neutral pH, such asalkylaminoethane, like diethylaminoethane (DEAE), dimethylaminoethane(DMAE), or trimethylaminoethyl (TMAE), polyethyleneimine (PEI),quaternary aminoalkyl, quaternary aminoethane (QAE), quaternary ammonium(Q), and the like.

In step (1), reducing agent is added to a concentration of 1 mM. Thedilute reducing agent used in step (2) is preferably at a concentrationof 1 μM. Both concentrations are for TCEP, and other values may apply toother reducing agents. The dilute reducing agent is used to maintain thepolypeptide in a reduced state prior to reaction with the molecularscaffold. Preferably, a chelating agent is included in the washing step.For example, EDTA may be included.

Alternative reducing agents may be selected from dithiothreitol,thioglycolic acid, thiolactic acid, 3-mercaptopropionic acid, thiomalicacid, 2,3-dimercaptosuccinic acid, cysteine, N-glycyi-L-cysteine,L-cysteinylglycine and also esters and salts thereof, thioglycerol,cysteamine and C1-C4 acyl derivatives thereof, N-mesylcysteamine,Nacetylcysteine, N-mercaptoalkylamides of sugars such asN-(mercapto-2-ethyl) gluconamide, pantetheine,N-(mercaptoalkyl)-co-hydroxyalkylamides, for example those described inpatent application EP-A-354 835, N-mono- orN,N-dialkylmercapto-4-butyramides, for example those described in patentapplication EP-A-368 763, aminomercaptoalkyl amides, for example thosedescribed in patent application EP-A-432 000,N-(mercaptoalkyl)succinamic acids and N-(mercaptoalkyl)succinimides, forexample those described in patent application EP-A-465 342, alkylaminemercaptoalkyl amides, for example those described in patent applicationEP-A-514 282, the azeotropic mixture of 2-hydroxypropyl thioglycolateand of (2-hydroxy-1-methyl)ethyl thioglycolate as described in patentapplication FR-A-2 679 448, mercaptoalkylamino amides, for example thosedescribed in patent application FR-A-2 692 481, andN-mercaptoalkylalkanediamides, for example those described in patentapplication EP-A-653 202.

The conjugation of the molecular scaffold, in the case of TBMB and otherscaffolds whose reactive groups are thiol-reactive, is preferablyconducted in the presence of acetonitrile. The acetonitrile ispreferably at a final concentration of about 20%.

Alternative scaffolds to TBMB are discussed herein.

Unreacted molecular scaffold is removed from the phage by washing.Subsequently, phage can be eluted from the resin, and selected as setforth previously.

Additional steps can also be included in the procedure. Such steps arenot mandatory, and do not significantly increase the yield or efficiencyof the process.

For example, the phage-containing culture medium, combined with theresin, can be washed prior to reduction with the reducing agent. Thereducing agent itself can be added in two steps; in a concentrated form,to effect reduction, and then in dilute form (step 2 above), to maintainthe displayed polypeptide in a reduced state.

The timing of the steps can also be varied, without significantlyaltering the efficiency of the procedure. For example, the presentinvention found that reduction in TCEP for 20 minutes is as effective asreduction for 30 minutes. Likewise, reaction with TBMB for 10 minutesdoes not give a significantly lower level of binding than reaction for30 minutes.

(v) Magnetic Separation

In an advantageous embodiment, the resin is magnetic. This allows thepolypeptide-bearing phage to be isolated by magnetic separation.Magnetic resin beads, such as magnetic sepharose beads, can be obtainedcommercially from, for example, Bangs Laboratories, Invitrogen, Origeneand GE Healthcare. See also U.S. Pat. No. 2,642,514 and GB 1239978.Application of a magnetic field permits isolation of the beads, whichresults in purification of the polypeptides bound to the beads from themedium in which they are contained.

In one embodiment, the magnetic beads are separated from the medium byinsertion of a magnetic probe into the medium. Beads are retained on themagnetic probe, and can be transferred to a washing station, or adifferent medium. Alternatively, beads can be isolated by applying amagnetic field to the vessel in which they are contained, and removingthe medium once the beads are immobilised.

Magnetic separation provides faster, more efficient processing of resinsin the method of the invention.

(C) Use of Polypeptide Ligands According to the Invention

Polypeptide ligands selected according to the method of the presentinvention may be employed in in vivo therapeutic and prophylacticapplications, in vitro and in vivo diagnostic applications, in vitroassay and reagent applications, and the like. Ligands having selectedlevels of specificity are useful in applications which involve testingin non-human animals, where cross-reactivity is desirable, or indiagnostic applications, where cross-reactivity with homologues orparalogues needs to be carefully controlled. In some applications, suchas vaccine applications, the ability to elicit an immune response topredetermined ranges of antigens can be exploited to tailor a vaccine tospecific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity arepreferred for administration to a mammal, and 98 to 99% or morehomogeneity is most preferred for pharmaceutical uses, especially whenthe mammal is a human. Once purified, partially or to homogeneity asdesired, the selected polypeptides may be used diagnostically ortherapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings and the like(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes Iand II, Academic Press, NY).

The peptide ligands of the present invention will typically find use inpreventing, suppressing or treating inflammatory states, allergichypersensitivity, cancer, bacterial or viral infection, and autoimmunedisorders (which include, but are not limited to, Type I diabetes,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus,Crohn's disease and myasthenia gravis).

In the instant application, the term “prevention” involvesadministration of the protective composition prior to the induction ofthe disease. “Suppression” refers to administration of the compositionafter an inductive event, but prior to the clinical appearance of thedisease. “Treatment” involves administration of the protectivecomposition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe peptide ligands in protecting against or treating the disease areavailable. The use of animal model systems is facilitated by the presentinvention, which allows the development of polypeptide ligands which cancross react with human and animal targets, to allow the use of animalmodels.

Methods for the testing of systemic lupus erythematosus (SLE) insusceptible mice are known in the art (Knight et al. (1978) J Exp. Med.,147: 1653; Reinersten et al. (1978) New Eng. J: Med., 299: 515).Myasthenia Gravis (MG) is tested in SJL/J female mice by inducing thedisease with soluble AchR protein from another species (Lindstrom et al.(1988) Adv. Inzn7unol., 42: 233). Arthritis is induced in a susceptiblestrain of mice by injection of Type II collagen (Stuart et al. (1984)Ann. Rev. Immunol., 42: 233). A model by which adjuvant arthritis isinduced in susceptible rats by injection of mycobacterial heat shockprotein has been described (Van Eden et al. (1988) Nature, 331: 171).Thyroiditis is induced in mice by administration of thyroglobulin asdescribed (Maron et al. (1980) J. Exp. Med., 152: 1115). Insulindependent diabetes mellitus (IDDM) occurs naturally or can be induced incertain strains of mice such as those described by Kanasawa et al.(1984) Diabetologia, 27: 113. EAE in mouse and rat serves as a model forMS in human. In this model, the demyelinating disease is induced byadministration of myelin basic protein (see Paterson (1986) Textbook ofImmunopathology, Mischer et al., eds., Grune and Stratton, New York, pp.179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al.(1987) J. Immunol., 138: 179).

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate carriers. Typically, thesecarriers include aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, any including saline and/or buffered media. Parenteralvehicles include sodium chloride solution, Ringer's dextrose, dextroseand sodium chloride and lactated Ringer's. Suitablephysiologically-acceptable adjuvants, if necessary to keep a polypeptidecomplex in suspension, may be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention may be used as separatelyadministered compositions or in conjunction with other agents. These caninclude antibodies, antibody fragments and various immunotherapeuticdrugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum,and immunotoxins. Pharmaceutical compositions can include “cocktails” ofvarious cytotoxic or other agents in conjunction with the selectedantibodies, receptors or binding proteins thereof of the presentinvention, or even combinations of selected polypeptides according tothe present invention having different specificities, such aspolypeptides selected using different target ligands, whether or notthey are pooled prior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the selected antibodies, receptors or binding proteinsthereof of the invention can be administered to any patient inaccordance with standard techniques. The administration can be by anyappropriate mode, including parenterally, intravenously,intramuscularly, intraperitoneally, transdermally, via the pulmonaryroute, or also, appropriately, by direct infusion with a catheter. Thedosage and frequency of administration will depend on the age, sex andcondition of the patient, concurrent administration of other drugs,counterindications and other parameters to be taken into account by theclinician.

The peptide ligands of this invention can be lyophilised for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that use levels may have to beadjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktailthereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of selected peptide ligand per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. For prophylactic applications, compositions containing the presentpeptide ligands or cocktails thereof may also be administered in similaror slightly lower dosages.

A composition containing a peptide ligand according to the presentinvention may be utilised in prophylactic and therapeutic settings toaid in the alteration, inactivation, killing or removal of a selecttarget cell population in a mammal. In addition, the selectedrepertoires of polypeptides described herein may be usedextracorporeally or in vitro selectively to kill, deplete or otherwiseeffectively remove a target cell population from a heterogeneouscollection of cells. Blood from a mammal may be combinedextracorporeally with the selected peptide ligands whereby the undesiredcells are killed or otherwise removed from the blood for return to themammal in accordance with standard techniques.

(D) Mutation of Polypeptides

The desired diversity is typically generated by varying the selectedmolecule at one or more positions. The positions to be changed areselected, such that libraries are constructed for each individualposition in the loop sequences. Where appropriate, one or more positionsmay be omitted from the selection procedure, for instance if it becomesapparent that those positions are not available for mutation withoutloss of activity.

The variation can then be achieved either by randomisation, during whichthe resident amino acid is replaced by any amino acid or analoguethereof, natural or synthetic, producing a very large number of variantsor by replacing the resident amino acid with one or more of a definedsubset of amino acids, producing a more limited number of variants.

Various methods have been reported for introducing such diversity.Methods for mutating selected positions are also well known in the artand include the use of mismatched oligonucleotides or degenerateoligonucleotides, with or without the use of PCR. For example, severalsynthetic antibody libraries have been created by targeting mutations tothe antigen binding loops. The same techniques could be used in thecontext of the present invention. For example, the H3 region of a humantetanus toxoid-binding Fab has been randomised to create a range of newbinding specificities (Barbas et al. (1992) Proc. Natl. Acad. Sci. USA,89: 4457). Random or semi-random H3 and L3 regions have been appended togermline V gene segments to produce large libraries with mutatedframework regions (Hoogenboom- & Winter (1992) R Mol. Biol., 227: 381;Barbas et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 4457; Nissim et al.(1994) EMBO J, 13: 692; Griffiths et al. (1994) EMBO J, 13: 3245; DeKruif et al. (1995) J. Mol. Biol., 248: 97). Such diversification hasbeen extended to include some or all of the other antigen binding loops(Crameri et al. (1996) Nature Med., 2: 100; Riechmann et al. (1995)Bio/Technology, 13: 475; Morphosys, WO97/08320, supra).

However, since the polypeptides used in the present invention are muchsmaller than antibodies, the preferred method is to synthesise mutantpolypeptides de novo. Mutagenesis of structured polypeptides isdescribed above, in connection with library construction.

The invention is further described below with reference to the followingexamples.

EXAMPLES Comparative Example 1

This example is taken from WO2009/098450.

In this example, it was demonstrated that attaching phage displayedpeptides to small molecules. The polypeptide in this example is a phagedisplayed peptide. The nucleic acid is comprised by the phage particle.The molecular scaffold in this example is a small molecule (TBMB).

Typically 10¹¹-10¹² t.u. of PEG purified phage were reduced in 20 ml of20 mM NH4HCO₃, pH 8 with 1 mM TCEP at 42° C. for 1 hr. The phage werespun at 4000 rpm in a vivaspin-20 filter (MWCO of 10′000) to reduce thevolume of the reduction buffer to 1 ml and washed twice with 10 ml icecold reaction buffer (20 mM NH4HCO₃, 5 mM EDTA, pH 8). The volume of thereduced phage was adjusted to 32 ml with reaction buffer and 8 ml of 50μM TBMB in ACN were added to obtain a final TBMB concentration of 10 μM.The reaction was incubated at 30° C. for 1 hr before non-reacted TBMBwas removed by precipitation of the phage with 1/5 volume of 20% PEG,2.5 M NaCl on ice and centrifugation at 4000 rpm for 30 minutes.

The small organic compound tris-(bromomethyl)benzene (TBMB) was used asa scaffold to anchor peptides containing three cysteine residues (Kemp,D. S, and McNamara, P. E., J. Org. Chem, 1985; FIG. 1B). Halogen alkanesconjugated to an aromatic scaffold react specifically with thiol groupsof cysteines in aqueous solvent at room temperature (Stefanova, H. I.,Biochemistry, 1993). Meloen and co-workers had previously usedbromomethyl-substituted synthetic scaffolds for the immobilization ofpeptides with multiple cysteines (Timmerman, P. et al., ChemBioChem,2005). The mild conditions needed for the substitution reaction areconvenient to spare the functionality of the phage (Olofsson, L., etal., J. of Molecular Recognition, 1998). Cysteines were chosen asanchoring points because their side chains have the most distinguishedreactivity within the 20 natural amino acids. Also, cysteine residuesare rare in proteins of the phage coat (8 cysteines in plll, onecysteine in pVI, pVII and pIX; Petrenko, V. A. and Smith, G. P., PhageDisplay in Biotechnology and Drug Discovery, 2005). The three-foldrotational symmetry of the TBMB molecule ensures the formation of aunique structural and spatial isomer upon reaction with three cysteinesin a peptide.

The reaction conditions for the modification of a peptide on phage wereelaborated next. As it appeared difficult to detect the chemicallymodified peptide on phage with available techniques, the presentinvention expressed the peptide ^(N)GCGSGCGSGCG^(C) as an N-terminalfusion with the two soluble domains D1 and D2 of the minor phage coatprotein plll and analyzed the molecular weight of the protein before andafter reaction with TBMB by mass spectrometry. Attempts to selectivelylink the three cysteines in the peptide to the scaffold but spare thethree disulfide bridges of the D1 and D2 domains of plll (C7-C36,C46-053, C188-C201) failed. This prompted us to take advantage of adisulfide-free gene-3-protein recently developed by Schmidt F. X. andco-workers (Kather, I. et al., J. Mol. Biol., 2005). The peptide fusedto the N-terminal domain of the cysteine-free gill protein was reducedwith tris(carboxyethyl)phosphine (TCEP). As the reducing agent was foundto react with the bromomethyl groups of the TBMB scaffold, it wasremoved before the addition of TBMB to the protein. Re-oxidation of thethiol groups after removal of TCEP could be prevented by degassing ofthe reaction buffer and complexation of metal ions with 5 mM EDTA.Reaction of the thiol groups with TBMB at various concentrations andmass spectrometric analysis of the product revealed that a concentrationof 10 μM TBMB is sufficient for quantitative modification of the peptideat 30° C. in one hour. Predominantly one product was formed with theexpected molecular mass (Δ mass expected=114 Da; FIG. 1A). When thedisulfide-free D1-D2 without a fused peptide was incubated with TBMB,its mass was not changed indicating that non-specific reactions withother amino acids do not occur. Addition of phage particles to thereactions (10¹⁰ t.u. per millilitre) revealed that the high density ofphage coat proteins in the vessel does not encumber the reaction of thepeptide with TBMB. Unexpectedly, it was found that reaction of TBMB withpeptides containing only two cysteine residues(^(N)AGSGCGSGCG^(C)-D1-D2) yields a product with a molecular mass thatis consistent with the reaction of the remaining bromomethyl group withthe primary amine of the N-terminus (FIGS. 2A and 2B). Similarly, thereaction of TBMB with a peptide having one cysteine and a lysine(^(N)AGSGKGSGCG^(C)-D1-D2) yields a molecular mass that is expected whenthe primary amines of lysine and the N-terminus react with the remainingtwo bromomethyl groups (FIGS. 2C and 2D).

Example 2 Modification of Phage on Resin

PK15 is a three cysteines containing peptide (H-ACSDRFRNCPADEALCG-NH₂),which when coupled with TBMB, is a specific and potent inhibitor ofhuman plasma kallikrein. This peptide can be displayed as a fusion togene 3 protein of phage and if correctly modified by TBMB will result ina phage that can specifically bind to kallikrein. Non-modification ofPK15 on the phage or cross-linking of the phage would not result in aspecific binding signal for the phage binding to kallikrein.

Anion exchange resin was used to capture the phage, allowing for quickand easy changing of the buffers that the phage were exposed to duringthe modification process. The phage were also titred for particle numberand infectivity to show that the modification process had not made thephage significantly less infectious.

Materials and Methods

-   -   1. 1 ml of 1M NaHCO₃ was added to either 50 μl, 100 μl or 150 μl        of an approximately 50% slurry of a strong anion exchange resin        to equilibrated the resin.    -   2. Each sample was spun at 3000 rpm in a microfuge for one        minute before the supernatant was carefully removed.    -   3. 1 ml of overnight culture, from which the E. coli had been        removed by centrifugation, containing PK15 expressing phage was        added to each sample, followed by 10 μl of NaHCO₃ and 1 μl of 1M        TCEP. The NaHCO₃ was added to raise the pH of the solution to        allow the phage to bind to the resin and the TCEP is a reducing        agent. The samples were mixed by rotation for 20 minutes.    -   4. The samples were centrifuged as before and the supernatant        carefully removed.    -   5. 1 ml of 20 mM NaHCO₃, 5 mM EDTA containing 1 μM TCEP was        added to resuspend the resin whilst washing away the majority of        the any remaining TCEP prior to the addition of TBMB.    -   6. The samples were centrifuged and the supernatant carefully        removed.    -   7. 1 ml of 20% acetonitrile in 20 mM NaHCO₃, 5 mM EDTA        containing 60 μM TBMB was added to each sample. The samples were        mixed by rotation for 10 minutes.    -   8. The samples were centrifuged as before and the supernatant        carefully removed.    -   9. 1 ml of 20 mM NaHCO₃, 5 mM EDTA was added to each sample.    -   10. The samples were centrifuged as before and the supernatant        carefully removed.    -   11. 100 μl of 50 mM citrate pH 5.0, 1.5M NaCl was added to each        sample and the samples were mixed for 5 minutes on a shaking        platform.    -   12. Each sample was spun at 13000 rpm in a microfuge for one        minute before the supernatant was carefully removed and        retained. The supernatant was re-centrifuged, to remove any        remaining traces of the resin, and the supernatant was carefully        removed and retained.    -   13. Binding of the phage to kallikrein was performed.

The phage eluted from the resin bound specifically to kallikrein,demonstrating that the modification procedure had successfully createdTBMB coupled bicycle peptides on the phage (see FIG. 3).

Phage Titre

The particle and infectious titres for the samples were compared to seeif the modification procedure had “damaged” the phage and rendered themless infectious than before the modification.

Total phage titre per ml Particle Infective Ratio  50 μl resin 3.0E+112.80E+10 10.5 100 μl resin 2.9E+11 3.40E+10 8.6 150 μl resin 2.6E+112.80E+10 9.1

The roughly 10 fold higher particle titre than infective titre istypical of pre-modification ratio for phage in our laboratories usingthe standard procedures described above. The modification process hastherefore not significantly damaged the phage.

Example 3 Polypeptide Modification on Phage Using Magnetic Separation

The use of a magnetic separation station for the isolation of phagedisplaying polypeptides is described. In addition, in the presentexample, the effect of:

-   -   Different binding buffers (i.e. phage input solution)    -   Different binding/wash buffers (i.e. buffer during modification)    -   Different elution buffers        on the efficiency and yield of the magnetic TCEP/TBMB        modification process is reviewed. The polypeptide used was PK15,        displayed on wild-type FdTet.

Materials and Methods

A colony from E. coli containing PK15/WT FdTet which had been freshlystreaked on an agar plate was used to inoculate 25 ml of either 2TY/tetor LB/tet, and cultures were incubated overnight at 37 C shaking 250rpm.

The following solutions were prepared:

Elution Buffers:

Citrate solution=100 mM (2×)→pH2.0 (without adjustment)

20 ml portions of the 100 mM citrate buffer were diluted 1-in-2 withwater, then pH-adjusted (with NaOH) to

-   -   pH3.5    -   pH4    -   pH5

10 ml portions of each pH solution were supplemented with NaCl to

-   -   1M    -   1.5M    -   2M

Binding/Wash/Modification Buffers:

The buffers compared were NH₄CO₃ and NaHCO₃.

1M (50×) NaHCO₃ solution reaches pH9.0 (without adjustment)

20 mM NaHCO₃ buffer (using the 1M solution) and 5 mM EDTA added reachespH9.0 (without adjustment). The 20 mM NaHCO₃ buffer was degassed for 1hr.

Samples to be treated in NaHCO₃ buffer were prepared using 1M NaHCO₃.

The two PK15 cultures were treated as follows:

Measured OD600: PK15 in 2TY = 1.95 PK15 in LB = 2.056 Measured pH: PK15in 2TY = pH 8.5 PK15 in LB = pH 7.5 Either 1M Tris pH 8 (to 10 mM final)for samples in NH₄CO₃ buffer or 1M NaHCO₃ (to 20 mM final) for samplesin NaHCO₃ was added to the cultures and pH as measured: PK15/2TY/Tris =pH 8 PK15/2TY/NaHCO₃ = pH 9 PK15/LB/NaHCO₃ = pH 8

The following solutions at the specified pH were prepared.

1 ml NH₄CO₃/EDTA buffer pH 8 1 ml NH₄CO₃/EDTA buffer + 1 mM TCEP pH 7 1ml NH₄CO₃/EDTA buffer + 1 μM TCEP pH 7 800 μl NH₄CO₃/EDTA buffer + 199μl acetonitrile + pH 7 60 μM TBMB 1 ml NaHCO₃/EDTA buffer pH 8 1 mlNaHCO₃/EDTA buffer + 1 mM TCEP pH 7 1 ml NaHCO₃/EDTA buffer + 1 μM TCEPpH 7 800 μl NaHCO₃/EDTA buffer + 199 μl acetonitrile + pH 7 60 μM TBMB

Magnetic separation of the chromatography was performed, retainingeither the beads or the supernatant where appropriate

Part A:

For each sample 20 μl magnetic ion exchange beads were rinsed in 1 mlNH₄CO₃/EDTA buffer and were resuspended in 10 μl of the same buffer. Thesamples were then processed as follows

-   -   A. 980 μl phage solution+10 μl washed beads+10 μl 1M Tris pH8    -   B. The samples were mixed for 20 minutes before the beads were        magnetically separated from the solution and the beads were        retained.    -   C. The beads were washed with 1 ml of either NaHCO₃ or        NH4CO₃/EDTA buffer with 1 minute's mixing before capturing the        beads magnetically.    -   D. The beads were washed with 1 ml of either NaHCO₃ or        NH4CO₃/EDTA+1 mM TCEP buffer with 20 minutes mixing before        capturing the beads magnetically The beads were washed with 1 ml        of either NaHCO₃ or NH4CO₃/EDTA buffer+1 μM TCEP buffer with 1        minutes mixing before capturing the beads magnetically    -   E. The beads were then added to 800 μl either NaHCO₃ or        NH4CO₃/EDTA buffer+200 μl acetonitrile/300 μM TBMB (60 μM TBMB        final concentration) and allowed to mix for 30 minutes before        capturing the beads magnetically    -   F. The beads were washed with 1 ml of either NaHCO₃ or        NH4CO₃/EDTA buffer with 1 minute's mixing before capturing the        beads magnetically.    -   G. The beads were then added to 50 μl 50 mM citrate elution        buffer (pH 3.5/4/5; NaCl 1M/1.5M/2M) for 1 minute with mixing.    -   H. The beads were then captured magnetically and the supernatant        retained.

Finally, 10 μl of 1M Tris pH8 was added to the 50 μl eluate toneutralise it.

Samples performed:

Input (binding) Modification Elution # Media Phage Buffer Buffer pH[NaCl] 1 2TY Culture 10 mMTris pH8 NH₄CO₃/ 4 1.5 EDTA 2 2TY Supernatant10 mM Tris pH8 NH₄CO₃/ 4 1.5 EDTA 3 2TY Culture 20 mM NaHCO₃ NaHCO₃/ 41.5 pH9 EDTA 4 LB Culture 20 mM NaHCO₃ NaHCO₃/ 4 1.5 pH9 EDTA A 2TYCulture 20 mM NaHCO₃ NaHCO₃/ 4 1.5 pH9 EDTA B 2TY Culture 20 mM NaHCO₃NaHCO₃/ 3.5 1.5 pH9 EDTA C 2TY Culture 20 mM NaHCO₃ NaHCO₃/ 5 1.5 pH9EDTA D 2TY Culture 20 mM NaHCO₃ NaHCO₃/ 4 1 pH9 EDTA E 2TY Culture 20 mMNaHCO₃ NaHCO₃/ 3.5 1 pH9 EDTA F 2TY Culture 20 mM NaHCO₃ NaHCO₃/ 5 1 pH9EDTA G 2TY Culture 20 mM NaHCO₃ NaHCO₃/ 4 2 pH9 EDTA H 2TY Culture 20 mMNaHCO₃ NaHCO₃/ 3.5 2 pH9 EDTA I 2TY Culture 20 mM NaHCO₃ NaHCO₃/ 5 2 pH9EDTA

The phage eluates (in citrate buffer) were retained.

In order to see whether each different elution buffer has left anynon-eluted phage bound to the beads, a second elution was performedusing the same elution buffer.

Samples were assayed by qPCR for particle titre. The results are shownin FIG. 4.

Conclusions:

-   -   The nature of the culture media (2TY or LB) does not        significantly affect the input phage titre (the 2-fold        difference seen is probably within the variability of the qPCR        assay).    -   The nature of the binding buffer (Tris or NaHCO₃) does not        significantly affect the number of eluted phage    -   The nature of the wash/modification buffer does not        significantly affect the number of eluted phage    -   For all types of input/wash eluted in pH4 1.5M buffer as usual,        30-40% of the input phage are eluted following modification.    -   There are no clear trends regarding which pH or [NaCl] is best        for elution, but:        -   pH3.5 elution buffer is generally poor        -   2M NaCl is generally poor    -   An elution buffer that elutes efficiently in the 1^(st) elution,        generally elutes well in the 2^(nd) elution (even if there may        be expected to be less phage retained on the beads after the        1^(st) elution).

In order to check the modification of the above samples, the elutedphage were screened for Kallikrein binding.

A target binding screen was performed on the eluate samples from above,as shown in FIG. 5. No clear trends were visible, even on repeat assays.

Analysis of Elution Buffers

The binding/elution and modification procedure was repeated withdifferent elution buffers.

Samples performed as set forth below:

Input (binding) Modification Elution # Media Phage Buffer Buffer pH[NaCl] 2 2TY Supernatant 10 mM Tris pH8 NH₄CO₃/ 4 1.5 EDTA 3 2TYSupernatant 20 mM NaHCO₃ NH₄CO₃/ 4 1.5 pH9 EDTA 4 LB Supernatant 20 mMNaHCO₃ NaHCO₃/ 4 1.5 pH9 EDTA A 2TY Supernatant 20 mM NaHCO₃ NaHCO₃/ 41.5 pH9 EDTA B 2TY Supernatant 20 mM NaHCO₃ NaHCO₃/ 3.5 1.5 pH9 EDTA C2TY Supernatant 20 mM NaHCO₃ NaHCO₃/ 5 1.5 pH9 EDTA D 2TY Supernatant 20mM NaHCO₃ NaHCO₃/ 4 1 pH9 EDTA E 2TY Supernatant 20 mM NaHCO₃ NaHCO₃/3.5 1 pH9 EDTA F 2TY Supernatant 20 mM NaHCO₃ NaHCO₃/ 5 1 pH9 EDTA G 2TYSupernatant 20 mM NaHCO₃ NaHCO₃/ 4 2 pH9 EDTA H 2TY Supernatant 20 mMNaHCO₃ NaHCO₃/ 3.5 2 pH9 EDTA I 2TY Supernatant 20 mM NaHCO₃ NaHCO₃/ 5 2pH9 EDTA

Conclusions:

A trend is seen between the eluates from different input samples:

NH₄CO₃/Tris/TY<NaHCO₃/NaHCO₃/TY<NaHCO₃/NaHCO₃/LB

However the difference between these does not appear to be significant.

Likewise, a trend is seen between the eluates using different elutionbuffers:

-   -   pH 3.5 gives poor elution irrespective of [NaCl]    -   pH 5 gives best elution when using 1.5M or 2M NaCl    -   pH 4 gives good elution at lower salt

pH5 gives the best results, but must be used with high salt.

Example 4 Comparison of ‘Quick’ and ‘Long’ Magnetic Phage ModificationProtocols

The phage modification process has been optimised from a ‘long’protocol. The results of the long protocol are compared herein to ashortened protocol.

A colony from streaked PK15/WT FdTet plate as in Example 3 was used toinoculate 25 ml of 2TY/tet. The culture incubated overnight at 37° C.,shaking at 250 rpm.

Long and quick protocols were performed. These are illustrated in FIG.6.

The quick protocol is as follows

-   -   Rinse 20 μl magnetic ion exchange beads in 1 ml 1M NaHCO₃ buffer        and resuspend in 10 μl of the same buffer.        -   A. 1 ml input solution (Culture/beads/TCEP), mix for 20            minutes and capture the beads magnetically.        -   B. Wash the beads in 1 ml NaHCO₃/EDTA buffer+1 μM TCEP by            mixing the beads with the buffer and immediately recapturing            the beads magnetically        -   C. Mix the beads in NaHCO₃/EDTA buffer+(TBMB in ACN)    -   where [ACN]_(final)=20%; [TBMB]_(final)=60 μM for 10 minutes        before capturing the beads magnetically        -   D. Wash the beads in 1 ml NaHCO₃/EDTA buffer by mixing the            beads with the buffer and immediately recapturing the beads            magnetically        -   E. Elute the phage from the beads by mixing with 50 μl 50 mM            citrate 1.5M NaCl pH5 for 1 minute before magnetically            capturing the beads nad retaining the supernatant.    -   Finally, 10 μl of 1M Tris pH8 was added to the 50 μl eluate to        neutralise it.

The long protocol is as follows:

Part A:

-   -   Rinse 20 μl magnetic ion exchange beads in 1 ml 1M NaHCO₃ and        resuspend in 10 μl of the same buffer.        -   A. 980 μl phage solution+10 μl washed beads+10 μl 1M NaHCO₃            mix for 20 minutes and capture the beads magnetically.        -   B. Wash the beads in 1 ml NaHCO₃/EDTA buffer by mixing the            beads with the buffer and immediately recapturing the beads            magnetically        -   C. Mix the beads in 1 ml NaHCO₃/EDTA buffer ±1 mM TCEP for            30 minutes and capture the beads magnetically.        -   D. Wash the beads in 1 ml NaHCO₃/EDTA buffer ±1 μM TCEP by            mixing the beads with the buffer and immediately recapturing            the beads magnetically        -   E. Mix the beads in NaHCO₃/EDTA buffer+(TBMB in ACN)            -   where [ACN]_(final)=20%; [TBMB]_(final)=60 μM for 30                minutes and capture the beads magnetically.        -   D. Wash the beads in 1 ml NaHCO₃/EDTA buffer by mixing the            beads with the buffer and immediately recapturing the beads            magnetically        -   C. Elute the phage from the beads by mixing with 50 μl 50 mM            citrate 1.5M NaCl pH5 for 1 minute before magnetically            capturing the beads and retaining the supernatant.    -   Finally, 10 μl of 1M Tris pH8 was added to the 50 μl eluate to        neutralise it.

Samples where TCEP and TBMB had been omitted (‘Non-modified’) wereincluded.

The infective titre of output samples and input (culture supernatant)was assayed as follows:

-   -   E. coli HB2151 were grown and aliquoted in 2YT until OD600=˜0.5

This represents ˜2.5×10̂8 cells/ml

-   -   Phage samples were diluted 1 in 1000 in 2YT    -   1 μl of diluted samples was added to 1 ml HB2151 (2.5×10̂8 cells)    -   The sample was incubated for 1 hr 37 C shaking 250 rpm    -   7×10-fold serial dilutions were made (neat→10̂−7) in 2YT    -   20 μl of each was spotted onto dried tetracycline agar plates        and incubated overnight at 37 C    -   The samples were analysed by qPCR. Results are shown in FIG. 7A.

A Kallikrein-binding assay was carried out on the samples in order tocheck for successful cyclisation. The results are shown in FIG. 7B.

Conclusions:

-   -   The ‘Quick’ and ‘Long’ protocols produce modified phage which        give similar levels of signal in a kallikrein-binding assay    -   Use of the ‘Long’ protocol is more harmful to the infectivity of        the phage than the ‘Quick’ protocol. The quick protocol retains        the ˜1-in-10 infective phage as seen in the input; the long        protocol reduces the infectivity to 1-in-100.    -   Part of the phage damage seen in the long protocol can be        attributed to the longer manipulation time (i.e. the ‘long        protocol non-modified sample shows some loss of infectivity).

Overall, use of the new ‘Quick’ phage modification process leads to goodcyclisation without losing infectivity.

Unless otherwise stated, any methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention. Methods, devices, and materials suitable for suchuses are described above.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove embodiments is not to be limited to particular details set forthin the above description as many apparent variations thereof arepossible without departing from the spirit or scope of the presentinvention.

What is claimed is:
 1. A method for conjugating a peptide displayed on agenetic display system to a molecular scaffold, comprising the steps of:(a) combining polypeptides displayed on a genetic display system with apurification resin such that the display system is bound to the resinand treating the bound display system with a reducing agent; (b)exposing the bound display system to the molecular scaffold; (c)removing unreacted molecular scaffold from the bound display system; and(d) eluting the display system from the purification resin.
 2. Themethod according to claim 1, wherein the display system is phagedisplay.
 3. The method according to claim 1, wherein step (a) isfollowed by a washing step before addition of the molecular scaffold 4.The method according to claim 3, wherein the display system is washed ina dilute solution of reducing agent.
 5. The method according to claim 4,wherein the wash solution further comprises a chelating agent.
 6. Themethod according to claim 1, wherein the reducing agent is TCEP.
 7. Themethod according to claim 1, wherein the scaffold is TBMB.
 8. The methodaccording to claim 1, wherein the molecular scaffold is added in thepresence of aqueous acetonitrile.
 9. The method according to claim 1,wherein the resin is an anion exchange resin.
 10. The method accordingto claim 1, wherein the resin is magnetic.
 11. The method according toclaim 1, wherein step (a) is performed for 20 minutes at roomtemperature.
 12. The method according to claim 1, wherein step (b) isperformed for 10 minutes at room temperature.